Ergot preparation and process of obtaining same



Patented Oct. 6, 1936 UNITED- STAT S 2,056,360 PATENT OFFICE ERGOTPREPARATION AND PROCESS OF OBTAINING SAME Adelia McCrea, Detroit, Micln,assignor to Parke,

Davis & Company, Detroit, Mich a corporation of Michigan No Drawing.Application January 4, 1933, Serial No. 650,142

8 Claims. (01. 161-07) The invention relates to the artificialproduction of a drug useful as a substitute for the ergot oi' 10natural-ergot in certain essential characteristics.

but difiering from any other substance heretofore known.

A further object is to obtain a new process for manufacture of apharmaceutical product of the gilt type without requiring any naturallygrown These and other objects are obtained as a result oi my researchwhich has demonstrated for the first time that the fungus Clavicepspurpurea deveiops in saprophytic culture the three chief activeprinciples which are characteristic of the extracts made from thenatural sclerotia; viz, ergotoxin,

histamine, and tyramine; and that they are-obtainable to an extentsumciently large to be of economic significance.

The prior literature reveals the fact that Clam'ceps purpurea is afungus which attacks the naturally growing rye and develops the materialwhich is used commercially on a. large scale for the manufacture ofergot extracts. The prior literature also reveals the fact that thefungus can be cultured upon certain laboratory media but there isnothing prior to my research which has convincingly demonstrated thepossibility of producing on a commercial scale an artificially preparedsubstance useful as a substitute for the natural ergot of commerce.

I have discovered that by properly controlling the surroundingconditions it is possible to samephytically grow the fungus Claoicepspurpurea and obtain a material containing the chief characteristics ofthe natural ergot product including the particular alkaloids ergotoxineand ergotamine. In carrying out my process it is necessary to am obtaina culture of the fungus, then preferably to develop a single spore"strainand finally to transplant the strain'on a suitable medium whichwill cause sumcientdevelcpmentto pro-= vide af'final product having thedesired characterlsties. a r

The cultures oi the fungus may be bbtalned' in several ways! I U i. Fromthe ascosporea (perfect stage) of germinating sclerotta.

2. From the conidia (imperfect or "sphacelial" stage, the honeydew? ofrye infection).

3. From bits of tissue taken from. the inner portion oi matured (butliving) aclerotia, the ergot of commerce.

The first two methods are of course strictly seasonal due to the factthat they depend upon the growing rye, while the last method can be usedat any time when living sclerotia can be obtained. Since it is possibleto secure such sclerotia alive and active in the field each summer, itwill 5 be seen that by the third method cultures of the fungus arealways available.

By whatever procedure the original culture is obtained the subsequentsteps of the process are the same. Ai'ter isolation has given a purecul- 10 ture, it is advisable to develop a single spore strain fromwhich all .iuture plantings are then made although, so far as known,Clavioeps par purea. is homothallic. This pure, single spore strain isthen grown saprophytlcally (artificial- 15 1y) on any suitable mediumeither of liquid composition or of moist cereal composition, e. g.bran.- or ground rye. One formula that has been found to give goodgrowth contains the following inredients:

A Magnesium sulphate grams 0.625 Peptone do 0.625 Dihydrogen p o t a ssi u m phosphate ....grams 1.25 25 Maltese do 6.25 Malt extract do..-6.25 Water, distilled -s. cc. 1000 Other media capable of sustaininggrowth oi D Rye bran mixed with thirty per cent distilled water in whichone per cent asparasin is dissolved. 60 'When the cereal media are used,e. g.. round rye, rye bran, etc., growth is allowed to proceed as far aspossible in order that the material may be almost completely utilized bythe fungus. The 55 ,7

mass is then dried, ground if necessary, extracted and tested exactly asfor crude drug.

When a liquid medium is seeded with a spore suspension of the fungus,a'heavy ring of growth develops at the margin of the flasks. andscattered m discrete colonies form over the surface of the liquid whichlater join to make a mat of varying thickness according to the availablenutrients and the length of time allowed for growth. At least threeweeks should be given, better four, to permit full maturity but thisvaries somewhat with the food.

Cultures are then combined, pressed with a spatula. to free from excessmedium; left in petroleum ether several hours, drained on filter paperand rendered brittle by vacuum drying. Thismaterial is then ground,extracted, and tested exactly as for crude drug.

This fungus is not unduly exacting so far as physical factors areconcerned. Ordinary room temperature is suitable, as it has an optimalrange of 20 to 28 C., but a temperature range of from 18 to 30 C. can beemployed. Outside of these ranges the results are ordinarily not as goodalthough the organism is not completely destroyed attemperatures whichare considerably outside of this range. An abundant moisture supply mustbe available to permit normal vegatative growth before drying checksfurther development. Light exerts a marked effect upon the production ofcolor which appears to be close- 1y associated with the production ofergosterol, but it has not appeared that this factor increases thecontent or activity of alkaloids. Ample aeration is a requisite for goodgrowth. Oxygenation is a very definite stimulation to more rapidmetabolism and increased growth, provided that other factors are kept atoptimal conditions. The time necessary for securing what may be termednature mycelial mats corresponds approximately to the period of growthin rye heads under natural conditions, i. e., about a month from date ofinoculation.

When properly isolated and grown under suitable conditions, thissaprophytic fungus growth develops the characteristic activeprinciples, 1. e., the alkaloid constituents of ergot sclerotia grownnaturally as a parasite of rye. This has been repeatedly shown by teststhat conform strictly to themethods of the U. S. P. X. Extracts of thismaterial, therefore, exhibit the physiological activity and medicinalvalue of extracts made from the crude drug sclerotia, the ergot ofcommerce.

The mycelial mats hereinbefore described are not identical with naturalergot of commerce. One difference is that the mycelia of saprophyticallygrown Claviceps purpurea when germinated pass through the conidial(non-sexual) stage but do not pass through the ascospore (sexual) stageand consequently do not develop sclerotia. Thus the mycelial matsobtained by my invention not only ditfer from natural ergot in physicalform and appearance but also possess a real biological difference. Mynew product may be designated as non-sclerotial mycelium ofsaprophytically grown Claviceps purpurea. What I claim as my inventionis:

. 1. The process of obtaining a drug useful for the manufacture of ergotpreparations comprising the inoculation of a cereal culture mediumcontaining abundant moisture supply with a culture, of Clavicepspurpurea, maintaining said culture medium at a temperature of 20 to 28C.

' for a period of at least three or four weeks, drying the cerealmediifm and comminuting to obtain a series of particles adapted forextraction.

2. The process of manufacturing a pharmaceutical preparation comprisingthe placing of a cereal composition in a suitable container, said cerealmixture having an abundant supply of water, inoculating said cerealcomposition with a pure culture of C'laviceps purpurea, maintaining saidculture at a temperature of 20 to 28 C. for a time not less than threeweeks, removing the mass from the container, drying, comminuting andextracting the comminuted material to obtain a product havingphysiological properties characteristic of ergot.

3. The process of obtaining a product having the physiologicalproperties characteristic of natural ergot comprising the inoculationwith Claviceps purpurea of a culture medium comprising magnesiumsulphate, peptone, dihydrogen potassium phosphate, maltose, maltextract, and watergcontinuing the incubation at a temperature of 20 to28 C. until said culture medium is substantially utilized by the saidfungus, drying and comminuting the resulting mass, and extracting thesame to obtain a product having the alkaloids and thephysiological-properties characteristic of natural ergot.

4. The process of obtaining a product having the physiologicalproperties characteristic of natural ergot comprising the inoculation ofa culture medium having an abundant moisture supply with Clamcepspurpurea, maintaining said culture medium at a temperature within therange substantially from 18 C. to 30 C. for a period of time on theorder of three weeks or more suflicient to develop a mycelial mat andextracting said mycelial mat to obtain. a product having physiologicalproperties characteristic of extracts of natural ergot.

5. The process of obtaining a drug useful in the I 6. A drug comprisinga saprophytic growth of Claviceps purpurea, being in the form of a driedmycelial mat containing in extractable form alkaloids havingthephysiological characteristics of ergotoxin and ergotamine, saidmycelial mat being a vegetative growth free from sclerotia.

'7. A drug comprising a saprophytic growth of Claviceps purpureacontaining extractable alkaloids having physiological propertiescharacteristic of .the alkaloids of natural ergot, said growth beingvegetative and freefrom sclerotia.

.8. The process of obtaining a product having the physiologicalproperties characteristic of natural ergot comprising the inoculation ofa culture medium having an abundant moisture supply with Clamcepspurpurea, maintaining said culture medium'at a temperature within therange substantially from 18 C. to 30 C. for a period of time sufiicientto develop a mycelial mat and ex-

